Five dyes, acridine orange (AO), malachite green (MG), crystal violet (CV), congo red (CR) and methyl orange (MO) were used in this experiment. Various parameters of different light intensity such as visible, ultraviolet, sunlight and dark condition are observed for the de-coloration of these dyes. The de-coloration phenomenon was proceeded through adsorption assisted phot-degradation. The low cost, abundant nature, good recyclability and better dye removal efficiency make these catalysts suitable candidates for the de-coloration and mineralization of organic dyes.Cd-Al/C layered double hydroxide (Cd-Al/C-LDH) and Cd-Sb/C nanocatalyst are reported here for the de-coloration and mineralization of organic dyes. These catalysts were largely characterized by FESEM, EDS, XRD, FTIR, XPS, PL and DRS. The diffuse reflectance data showed a band gap at 2.92 and 2.983 eV for Cd-Al/C-LDH and Cd-Sb/C respectively. The band gap suggested that both catalysts work well in visible range. The photoluminescence spectra indicated a peak at 623 nm for both the catalysts which further support the effectiveness of the respective catalyst in visible range. Both catalysts also showed good recyclability and durability till 4 The highest toxicity were found in diazo and basic dyesDyes industries play an important role in the progress and development of a country and make the human life beautiful. In pre-historical time people used various dyes to make their environment gorgeous. Most of the dyes stuff are categorized on the basis of its coloring properties, solubility, and chemical nature232 and some inorganic ions. During the removal of dyes the AOPs generated highly reactive species known as ROS , which possibly invade almost all contaminants. In all known AOPs, the heterogeneous photocatalytic process is the most effective method because of its high availability, low toxicity, inexpensive and diverse nature that might attacked and mineralize a large number of contaminantsDyes removal from water is one of main environmental problem due to its carcinogenic and mutagenic nature to aquatic life11127141516172, in which divalent Mg+2 is octahedraly sandwich between hydroxyl groups6II1−x MIIIx(OH)2]z+(An−)z/n·yH2O MII and MIII are di- and trivalent cation,  while An− is the counter anion for cation present in the interlayer of brucite like sheets while x is the (MII/MII + MIII) ratio.A class of new materials with hydrotalcite like structure known as layered double hydroxide recently attract researchers attention due to their varied application and high anion exchange capacity which make them suitable candidates for catalysiset al. reported Mg-Al-CO3−2 for the de-coloration of indigo carmineet al. reported Mg/Al-LDH and study its various factor on its growth such as various metal cation concentration, pH and aging timeet al. investigated ZnTi-LDH by co-precipitation methods for the de-coloration of methylene blue dyes under the influence of visible light irradiationet al. through hydrothermal methodset al. as a support for NiAl-LDH through solution method2 adsorptionA number of authors have been reported various metal-metal combinations for the synthesis of layered double hydroxide through various techniques for various application. For instance, Gaini Salt of Al and cadmium nitrate and chloride of cadmium and antimony along with other reagents and dyes mentioned in this manuscript were obtained from Sigma-Aldrich, Ireland. Millipore-Q machine was used for double distilled water, present in chemistry department, King Abdulaziz University Saudi Arabia.3)3 and Cd(NO3)2 were dissolved thoroughly in double distilled water in 1:3 molar ratio. To this reaction mixture, 10 wt% of activated carbon was added and well dispersed by continuous stirring with the help of magnetic stirrer. To this mixture freshly prepared 0.1 M NaOH solution are added and continuously monitored till pH 9. After this the reaction mixture were placed on a hot plate for 6 h at 60 °C with homogenous stirring. After completion of the reaction the surplus solution is removed and the precipitate was washed thrice with C2H5OH:H2O mixture (8:2). The resultant product was dried in an oven for overnight at 50 °C and store in clean tube for further characterization.Salt of Cd and Al nitrate were well mixed in double distilled water and then mixed with activated carbon through co-precipitation method19282 and CdCl2.The Cd-Sb/C was synthesized in the same way as discuss above for Cd-Al/C-LDH by using SbClα radiations (λ = 0.154 nm) source were used for the purity and crystallinity of the catalyst. Field emission-scanning electron microscope (FESEM), JEOL  for surface morphology and average size of the particles, while, energy dispersive X-rays spectrometry (EDS) of oxford-EDS system was employed for the elemental composition of the catalyst. X-ray photoelectron spectroscopy (XPS) Thermo Scientific K-Alpha KA1066 spectrometer (Germany) in the range of 0 to 1350 eV was investigated for the elemental analysis as well as for the determination of binding energy in the respective catalyst. The photocatalytic reaction was monitored through Evolution 300 UV–visible spectrophotometer (Thermo scientific). The effect of visible and ultraviolet light on adsorption-degradation process of dyes were observed under visible lamp  and ultraviolet lamp  respectively. The solar light effect was studied under normal day sunlight in open atmosphere and dark effect in complete absence of light. Photoluminescence emission spectra were confirmed at 320 nm excitation wavelength (fluorescence spectrofluorophotometer), RF-5301 PC, Shimadzu, Japan. The UV–vis diffuse reflectance spectroscopy was recorded by PerkinElmer UV–vis diffuse reflectance spectrophotometer.FTIR (Thermo Scientific) for functional group analysis and powder X-ray diffractometer (PXRD) with a KFor this study 0.025% mmol effluent solution of acridine orange (AO), methyl orange (MO), congo red (CR), malachite green (MG) and crystal violet (CV) were prepared. The catalytic activity of Cd-Al/C-LDH and Cd-Sb/C were evaluated against the respective dyes under visible, solar, ultraviolet light, and dark condition. The adjusted dose 10 mg of the respective catalyst were added in 100 mL of 0.025 mmol concentration of dye solution and the gradual decrease in concentration of all the dyes were explored through UV-vis spectrophotometer. The % removal efficiency A.E. (%) of each catalyst was evaluated by using the following equation.0 represents the original concentration of each dye solution at time = 0, Ct is the concentration of dye solution by adding the catalyst after some time = t as indicated in equation (1). Similarly, A0 designated the absorbance of the original concentration of the dye solution at time = 0 and At is the absorbance of dye solution during reaction progress after passing some time = t. Each time 3 mL of the aliquot were taken after specified time and checked the reaction progress by using UV-vis. spectrophotometer.CThe average size and morphology was scrutinize through FESEM. The FESEM images shows the sheet morphology of Cd-Al/C-LDH  and Cd-S−1 which were attributed to the M–O, M–O–M and O–M–O bond in the lattice structure of metals of Cd-Al/C-LDH and Cd-Sb/C catalyst−1 recommending the presence of NO3−1 ions in the Cd-Al/C-LDH. The peak1402 at cm−1 confirmed that the synthesized catalyst were grown in layered double hydroxide (LDH) morphology. The NO3−1 ions were present in the interlayer structures of Cd-Al/C-LDH and Cd-Sb/C. Absorption peak at 3520 cm−1 exhibited in Cd-Al/C-LDH suggesting the OH stretching vibration in the brucite like layers (LDH). The broadening of this peak suggesting the presence of hydrogen bonding prompted by water molecule present in the interlayer of these hydrocalcite morphology−1 further suggesting the LDH nature of the respective catalyst. It was concluded from the FTIR spectrum that Cd-Al/C-LDH was grown in mixed metal oxide and LDH morphology.The functional groups in the respective catalyst were characterized through FTIR spectroscopy as indicated in 3−1 anions in the brucite like layers of Cd-Al/C-LDH. The characteristic peak for Cd-Al/C-LDH appeared at 2θ = 10.8 (003) which was not found in Cd-Sb/C. The 2θ = 23.4 (006) and 2θ = 34.4 (012) suggesting the formation of Cd-Al/C-LDH. The 003 and 006 corresponding to the basal reflection of the successive stacking of brucite like layers3−1 anions intercalated in the inner layer of LDH. The remaining XRD peaks correspond to the presence of Cd-Al doped oxide in the Cd-Al/C-LDH. Similarly, the basal peak at 2θ = 31.8 (111), 38.7 (200), 53.1 (220) indicating the formation of CdO while the remaining peaks are due to Sb2O3 in Cd-Sb/C. The XRD diffraction pattern shown that Cd-Al/C has grown in mixed oxide and LDH nature while Cd-Sb/C only in the form of oxide. The XRD data suggesting the intercalation of NO= 23.4 00 and 2θ =The composition analysis and binding energy of Cd-Al/C-LDH and Cd-Sb/C were determined by X-ray photo electron spectroscopy (XPS) from 10–1200 eV as depicts in The DRS spectra exhibited band gap at 2.92 eV for Cd-Al/C-LDH and 2.983 for Cd-Sb/C. The DRS data indicate that both catalyst work efficiently in the visible range as shown in The photocatalytic activity of the Cd-Al/C-LDH and Cd-Sb/C was carried out for the de-coloration of five dyes AO, CR, MO, MG and CV under solar, visible, ultraviolet light and dark condition.Prior to the effect of solar, ultraviolet and dark condition the effect of visible light was study on the de-coloration of cationic and anionic dyes. Both Cd-Al/C-LDH and Cd-Sb/C were evaluated against three different dyes cationic acridine orange (AO) and anionic congo red (CR) and methyl orange (MO) under visible of 400 watt.The catalytic dose was adjusted under visible light by selecting AO dye. Initially, the catalytic doses were adjusted with AO by starting from 100 mg of the respective catalyst in 100 mL of 0.025 mmol of AO. The Cd-Al/C-LDH de-colorize 70% AO while Cd-Sb/C 53% in 1 h. The amount of both the catalyst were decreased to 40 mg in 100 mL of 0.025 mmol of AO in which 63% of AO is removed with Cd-Al/C-LDH and approximately 45% with Cd-Sb/C in 1 h. However, the removal efficiency of dyes is further increased as we increased the contact time. For instance, after 2.5 h under the same condition the % removal of AO with Cd-Al/C-LDH and Cd-Sb/C was approximately 80 and 63% respectively as shown in Using small amount of the catalyst to dye solution is eco-friendly and therefore, we selected 10 mg of the respective catalyst as an optimized amount for the further detail study. 10 mg of the respective catalyst were further used for the de-colorization of AO, CR and MO. After the first 15 min the % removal efficiency of Cd-Al/C-LDH against AO, CR and MO was 27.0, 13.7 and 4.8% respectively. However, at the same condition the Cd-Sb/C showed 30.7, 3.9 and 3.7% removal efficiency respectively. During the start of the reaction Cd-Sb/C showed slightly good response then Cd-Al/C-LDH. However, onward its activity was much lower than Cd-Al/C-LDH and this might be due to the LDH nature of Cd-Al/C-LDH. By increasing the contact time the % de-coloration of all dyes is increased and it was found that after 200 min, the Cd-Al/C-LDH showed strong response then Cd-Sb/C. For instance, after 200 min the % de-coloration of AO with Cd-Al/C-LDH is 69.4% as compared to Cd-Sb/C which was only 44.0%. It was found that both catalyst showed superior response against cationic dye (AO) as compared to anionic dye (CR and MO) and this is probably due to the large structure of these mentioned anionic dyes. It was also found that for all dyes Cd-Al/C-LDH showed superior performance than Cd-Sb/C. The reaction was also monitored without catalyst with almost no change in dye concentration under visible light, which shows that visible light itself has no role in the de-coloration phenomena. The decrease in the concentration and % removal efficiency of CR and MO are illustrated in Under the same condition both the catalyst were evaluated for the de-coloration of AO in normal day sunlight exposure. The adjusted dose 10 mg of both the catalyst were added in 100 mL of 0.025 mmol of AO solution. The Cd-Al/C-LDH showed faster and better response then Cd-Sb/C in sunlight exposure. After the first 15 min of experiment the Cd-Al/C-LDH showed 13.5 while Cd-Sb/C showed 18.5% de-coloration of AO. Similarly, after 180 min the % de-coloration of AO with Cd-Al/C-LDH increased from 13.5 to 82.2% and Cd-Sb/C from 18.5 to 76.0%. This inferred the better performance of both catalyst with the passage of time as shown in the inset of Under visible and solar light irradiation it was concluded that cationic dye AO is selectively removed with the corresponding catalyst. It was also confirmed that cationic dye AO is predominantly removed in solar light as compared to visible light and it could possibly be the combine effect of visible, UV and IR regions in solar light. Therefore, we studied two more cationic dyes malachite green (MG) and crystal violet (CV) under the same condition and with same catalyst in solar light.After 180 mins under the same condition Cd-Al/C-LDH showed 82, 72 and 52% removal efficiency against AO, MG and CV dyes respectively. Similarly, Cd-Sb/C indicating approximately 75, 41 and 15% removal efficiency against AO, MG and CV respectively. The decrease in original concentration and percent removal of MG and CV with Cd-Al/C-LDH and Cd-Sb/C are depicted in The catalytic activity of the respective catalysts were excellent, better and good for AO, MG and CV respectively. Cd-Al/C-LDH showed stronger catalytic activity over Cd-Sb/C catalyst and selectively de-colorized AO over MG and CV as presented in UV lamp  was used for the de-coloration of 0.025 mmol AO solution. Keeping the same condition as adjusted for visible and solar light, 10 mg of both the catalyst were used for the de-coloration of 100 mL of 0.025 mmol AO solution. After the first 15 min of exposure time the % removal efficiency of Cd-Al/C-LDH and Cd-Sb/C against AO was 8.5 and 7.5% respectively. However, after 200 min, the removal efficiency of AO (%) was increased to 36.3% with Cd-Al/C-LDH and 29.1% with Cd-Sb/C. Like visible and solar light the Cd-Al/C-LDH showing superior removal for AO as compared to Cd-Sb/C. The decrease in concentration and % removal efficiency of AO under ultraviolet light exposure are presented in the inset of Prior to solar and ultraviolet light the effect of dark was studied for both catalyst to know the adsorption or degradation phenomena. The adjusted dose 10 mg of the respective catalyst were put in a beaker containing 100 mL of 0.025 mmol AO solution by providing complete dark condition. After the first 15 min of the reaction progress the Cd-Al/C-LDH displayed 11.0% and Cd-Sb/C 10.9% removal efficiency. However, after 200 min, the Cd-Al/C-LDH was showing 31.0% and Cd-Sb/C 26.0% removal of AO as indicated in After the detailed study for the de-coloration of dyes  under solar, visible, ultraviolet light and absence of light, it was concluded that cationic dyes removed preferentially then anionic dyes. Among the cationic dyes  AO was predominantly removed by both catalyst in solar light. However, the removal efficiency of MG and CV are also good. It was inferred that AO adsorbed in the absences of light and adsorbed plus degraded in ultraviolet, visible and solar light. The adsorption process is triggered by the presence of activated carbon in the respective catalyst. The dyes is adsorbed on the catalyst and then degraded as the reaction proceeded.t/Co). This model was applied to compare the rate of Cd-Al/C-LDH and Cd-Sb/C in visible light by using different amount of the catalyst against the removal of AO. This equation also applied to investigate the rate of reaction in different parameters like dark, ultraviolet, visible and solar light. The rate of the reaction was determined by plotting lnCt/Co should be subscript verses time. The model showed the highest rate of Cd-Al/C-LDH then Cd-Sb/C in all conditions. The rate of reaction is directly related to the amount of catalyst. For instance, at 40 mg of Cd-Al/C-LDH the rate of reaction was 5.88 × 10−3 mol L−1 min−1 as compared to 4.92 × 10−3 mol L−1 min−1 obtained when 10 mg of the same catalyst was used under solar light. The same trends was observed in visible light. However, the rate of degradation is slow from solar light as inspected in The kinetics of the reaction was determined by applying pseudo first order kinetics  of AO under visible light of 400 watt. After one hour, the reaction was stopped and 3 mL of the aliquot was taken through a clean syringe and examined in UV-vis. spectrophotometer. After this the catalyst was separated through filtration process and the filtrate was washed thrice with acetone. The recovered washed catalyst (obtained from first run) was used in the next cycle for 100 mL of 0.025 mmol AO solution without drying or heating. The reaction mixture was again placed for 1 h under visible light exposure. Similarly, after one hour the reaction was stopped for monitoring the decrease in concentration and catalytic activity through UV-vis spectrophotometer. The catalyst is likewise separated carefully and washed thrice with acetone and used in the third cycle for 100 mL of 0.025 mmol AO. The same practice recurred four times for Cd-Al/C-LDH and Cd-Sb/C. In each cycle the reaction mixture was placed under visible light for one hour. The Cd-Al/C-LDH indicated 70% removal efficiency in the first cycle and decreases to 53, 30 and 21% in second, third and four cycle respectively. Similarly, Cd-Sb/C indicated 52% AO removal in the first cycle which was decreased to 40, 25 and 11% in second, third and fourth cycle respectively. It was also found that Cd-Al/C-LDH was remained active. However, Cd-Sb/C lost its activity after forth cycle. Both the catalyst showed good recyclability and durability as shown in the inset of The structural features of dyes play a significant role in dyes light. However, it is necessary to select specific materials according to the functional groups in dyes. Dyes degradation generally undergoes through breakage of various functional group i.e. cleavage of the aromatic, C–S bond breaking occurred between an aromatic ring and the sulphur of a sulphonyl group. Similarly, other functional group such as C–C, C–N and azo bond breakage will also happened during dyes degradationThe inexpensive and diverse morphology make layered double hydroxide a suitable candidate in the field of catalysis. Cd-Al/C-LDH and Cd-Sb/C were synthesized through co-precipitation method. Both the catalyst showed a narrow band gap which indicated its effectiveness in visible region. The PL also showed a peak at 623 nm for both catalyst which showed its efficiency in visible range. Both catalyst grown in nanosheet morphologies. The Cd-Al/C has LDH nature as confirmed from FTIR and XRD. The respective catalyst were used for the de-coloration and mineralization of organic dyes from the effluent under solar, ultraviolet, visible light and dark condition. The Cd-Al/C-LDH shown better catalytic activity in all conditions and this is probably due to its layered double hydroxide nature. The rate of reaction was determined from Langmuir isotherm indicating high rate of Cd-Al/C-LDH as compared to Cd-Sb/C. A predominate degradation and negligible adsorption was found in solar light and an equal percentage of degradation and adsorption were found in visible light. Similarly, adsorption was observed in dark condition and ultraviolet light. The Cd-Al/C-LDH and Cd-Sb/C showed good recyclability, durability and easy separation. These results showed the high activity and the ease in separation of the catalyst which are routinely encounter in nanocatalysis.How to cite this article: Khan, S. A. et al. Layered double hydroxide of Cd-Al/C for the Mineralization and De-coloration of Dyes in Solar and Visible Light Exposure. Sci. Rep.6, 35107; doi: 10.1038/srep35107 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Several reports have suggested that peripheral blood-based parameters are associated with host immunity response, which is an essential component of the pathogenesis and progression of cancer. The purpose of the present study was to identify the prognostic significance of various peripheral blood-based biomarkers and to determine the optimal cut-off value suitable for luminal breast cancer patients. We found that lymphocyte-to-monocyte ratio (LMR) was significant prognostic predictors. And the patients with a CEF regimen and LMR ratio ≥ 5.2 gained a good prognosis. This study suggested that the LMR could be regarded as an independent prognostic factor in luminal breast cancer patients. The elevated LMR level also had enhanced 5-fluorouracil sensitivity in luminal breast cancer patients. Breast cancer is a complex and heterogeneous disease, and is categorized into three major subtypes based on gene expression profiles: luminal, basal like, and human epidermal growth factor 2 (HER2) enriched, which exhibit distinct clinical features, responses to specific chemotherapy, and prognosis –3. DespiRecent reports suggested that the peripheral blood-based parameters, such as absolute monocyte count (AMC), absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-lymphocyte ratio (PLR), are associated with host immunity response –14. MoreTo the best of our knowledge, there are no comprehensive data available evaluating a set of peripheral blood-based biomarkers in luminal breast cancers. The purpose of the present study was to identify the prognostic significance of various peripheral blood-based biomarkers, and to determine the optimal cut-off value suitable for luminal breast cancer patients.Data from two hundred and fifty-nine patients were collected for the analysis. The characteristics of the enrolled patients are generalized in Table P=0.006)  curves and area under the curve (AUC) were used to determine the optimal cutoff points for the LMR, NLR, PLR, AMC, and ALC, based on their utility as markers for the clinical outcome of relapse, cancer-related death. Regarding the LMR in luminal breast cancer patients, 5.2 was identified as the optimal cutoff point for distinguishing good prognosis patients from poor prognosis patients  Figure . There w) Figure .9/L  and the mean counts of monocytes were 0.41×109/L    (Table The mean counts of lymphocytes were 1.76×10P=0.001)  in luminal breast cancer patients with a LMR ≥5.2 was significantly longer than those with a LMR <5.2 (P<0.05)  after surgery. In luminal breast cancer patients with a LMR ≥5.2, Kaplan–Meier survival curves demonstrated that the DFS in patients with the CEF regimen was significantly longer than in those with the TAC regimen  Figure .P=0.001], lymph node status , and chemotherapy  were significantly related to a higher risk of recurrence. Similarly, LMR and chemotherapy also showed a significant association with DFS by multivariate analysis.Results of univariate and multivariate analysis to identify prognostic factors for DFS in luminal breast cancer patients are shown in Table P<0.01 and P< 0.01, respectively), as shown in Figure The MCF-7 and T47D cells are both luminal breast cancer cells. FAS expressions in 5-fluorouracil (5-FU) treated MCF-7 and T47D cell lines were higher than that in paclitaxel treated cell lines . Specifically, 5-FU-induced ICD exerts its chemoimmunotherapeutic effects by recruiting innate or adaptive immune cells, enhancing cancer cell antigenicity, and releasing cytokine secretion –42. The In conclusion, our study identified an optimal cut-off for the LMR restricted to luminal subtype breast cancer. Using this LMR index, our results revealed that an elevated LMR could be regarded as an independent prognostic factor in luminal breast cancer patients. Moreover, our results indicated that the elevated LMR level enhanced the 5-FU sensitivity in luminal breast cancer patients. Therefore, the combination of lymphocyte-monocyte ratio and chemotherapy may be a promising strategy for anticancer treatments. As this was a retrospective and single-center study, further investigations need to be performed to validate our conclusion.2, day 1; epirubicin 60 mg/m2, day 1; and 5-FU 600 mg/m2, day 1; every 21 days for 6 cycles) or TAC . Depending on the lymph node and HER2 status of the breast cancer, patients were selectively given radiation and targeted therapy. Patients with positive ER or PR status received endocrine therapy (20 mg of tamoxifen per day) for 5 years. After surgery, every patient was followed up regularly until December 2014 or until death. Patients who developed disease relapse were confirmed by adequate diagnostic imaging modalities and pathology during the follow-ups.This study, including the procedures for patient enrollment and recruitment, was approved by the Institutional Review Board of Tumor Hospital of Harbin Medical University and informed consent was obtained from each participant. The breast cancer patients were recruited between January 1st, 2006, and December 30th, 2008. The inclusion criteria were no metastatic lesions before surgery, infiltrating ductal carcinoma, ER/PR positive, and complete clinical and pathologic data. All patients in this study underwent an adjuvant chemotherapy regimen consisting of CEF . The peripheral LMR was calculated as the ratio of absolute counts between lymphocytes and monocytes. The peripheral NLR was translated as the ratio of absolute counts between neutrophils and lymphocytes. The peripheral PLR was interpreted as the ratio of absolute counts between platelets and lymphocytes. The peripheral blood samples were obtained after breast cancer diagnosis and before any treatment. All of the enrolled patients had no hematologic disorders or acute infections, implying that the cell counts could demonstrate the normal baseline value.All breast cancer tissues were formalin fixed and paraffin embedded. The immunohistochemical analysis was executed as described previously . In brie2. The cells were subcultured on 6-well plates for further experiments. After a 24h incubation to allow cell attachment, paclitaxel (0.2μM) or 5-FU (20μg/ml) were added to cells for 6h.MCF-7 and T47D cells were grown in in Roswell Park Memorial 1640 medium  containing 10% fetal calf serum, 100 mg/ml streptomycin and 100 units/ml penicillin and incubated at 37°C supplemented with 5% of COTotal RNA was extracted with the TRIzol reagent and cDNA was synthesized using a Prime-Script RT reagent kit . The following PCR conditions were used: 1 cycle at 95°C for 10 min and then 40 cycles of 15 s at 95°C and 1 min at 60°C. Real-time PCR was performed on the ABI PRISM7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. The expression changes of the RNA was normalized to β-actin RNA. The sequences of PCR primers used were as follows: sense, 5′-GTGAGGGAAGCGGTTTACGA-3′and anti-sense, 5′- AGATGCCCAGCATGGTTGTT-3′ for Fas; sense, 5′-CCACGAAACTACCTTCAACTCC-3′ and anti-sense, 5′-GTGATCTCCTTCTGCATCCTGT-3′ for β-actin.DFS was calculated as the time between diagnosis and disease relapse, or death from breast cancer. To evaluate whether increased LMR, NLR, PLR, AMC, or ALC correlated with prognosis of patients with breast cancer, we chose patients with disease relapse or cancer-related death during the follow-ups and defined them as the poor prognosis group. Other patients were included in the good prognosis group. ROC curves, and AUC, were performed to further evaluate the prediction status of LMR, NLR, PLR, AMC, and ALC. Sensitivity and specificity were calculated by the optimal cutoff points on the ROC curves, which decided the maximum value (sensitivity + specificity −1) of the Youden index .P-values <0.05. All statistical analyses were performed using the IBM SPSS 20.0 software .The Chi-square or Fisher's exact tests were used for comparing clinicopathologic features between different LMR groups. Continuous variables, reported as lymphocyte and monocyte counts, were compared by the Wilcoxon rank-sum test. Risk factors of DFS were analyzed by univariate analysis with the log-rank test and multivariate analysis with the Cox proportional hazard model. The survival curves were performed by the Kaplan–Meier method and the comparisons between groups were assessed by the log-rank test. The results were showed as hazard ratios HRs and 95% CIs. The significance of the relative differential expression of FAS was determined by the two-tailed unpaired t test. Statistical significance was decided as


Results were compared between groups. Optical coherence tomography from diabetic patients was evaluated for central retinal thickness (CRT) and macular volume (MV). Mean vitreous VEGF-B concentration was higher in diabetic (18.82 ± 1.44 pg/mL) vs. non-diabetic patients (17.90 ± 0.32 pg/mL) (p = 0.006), and in proliferative diabetic retinopathy (PDR) (19.03 ± 1.52 pg/mL) vs. non-PDR (NPDR) patients (18.18 ±0.96 pg/mL) (p = 0.025). In diabetic retinopathy (DR) patients, correlation between VEGF-B and CRT (μm) was positive and moderate: sr = 0.441 (p ≤ 0.05) and the correlation between VEGF-B and MV (mm3) was positive and robust: sr = 0.716 (p ≤ 0.01). VEGF-B levels are overexpressed in vitreous of diabetic patients, and the levels are higher in developed stages of DR. Correlation results show that CRT and MV increase with increased levels of VEGF-B. Targeting VEGF-B inhibition may have therapeutic beneficial implications.Vascular endothelial growth factor B (VEGF-B) is one of the enigmatic members of the VEGF family. The knowledge gap about VEGF-B expression and how its levels are altered in diabetic eyes were the focus of this investigation that was addressed by comparing and correlating vitreous VEGF-B between diabetic and non-diabetic patients. VEGF-B levels were measured by enzyme-linked immunosorbent assay in vitreous samples ( Vascular endothelial growth factor A (VEGF-A) is the most studied member of the VEGF family and is an important regulator of angiogenesis and vascular permeability in physiological and pathological conditions. It induces proliferation and migration of endothelial cells and vascular permeability, resulting in angiogenesis in vivo . Aiello Besides VEGF-A, there are other members of this family that may be of relevance for ocular disease. In particular, vascular endothelial growth factor B (VEGF-B) has been described in cardiovascular disease, diabetes, and cancer; however, its function in ophthalmology remains poorly understood, as it is the most controversial molecule of the VEGF family ,6,7. VEG167 and diffusible VEGF-B186 [VEGF-B was discovered in 1996 as a VEGF homolog . It is eEGF-B186 , as seenEGF-B186 ,13,14. VEGF-B186 . PlataniFurthermore, Lyer  studied VEGF-B was studied mainly in animal models; however, the expression of VEGF-B in human eyes and the changes in its levels in certain eye diseases, such as diabetic retinopathy (DR), need to be elucidated. A better understanding of the molecular pathological events triggered by the increase in VEGF expression, and specifically in VEGF-B, may be of fundamental importance for the development of new treatments for these pathologies. This is because the anti-angiogenic therapy is particularly promising for the treatment of neovascular diseases. Although the anti-angiogenic properties of VEGF-B are poor, VEGF-B promotes cell survival through its binding to VEGFR-1 and NRP-1, playing an important role as an anti-apoptotic molecule. It also suppresses growth through VEGFR-1 when it acts as a decoy for VEGF-A ,19.This project aims to provide a better understanding of the VEGF-B expression in human vitreous humor and its relationship with DR. It may contribute to the development of improved targeted anti-VEGF therapies for VEGF-driven eye diseases, with a lower occurrence of serious adverse events and ultimately for better effective therapeutic interventions. Thus, the main objective of this study was the quantification and comparison of VEGF-B levels in vitreous samples of diabetic and non-diabetic patients. Additionally, VEGF-B levels were compared between patients with non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) and correlated with central retinal thickness (CRT) and macular volume (MV). In addition, the glucose levels in the vitreous were measured and compared between the study groups: diabetic versus non-diabetic patients and NPDR versus PDR.n = 19) or NPDR (n = 6) (13 women and 12 men) and a control group of 8 non-diabetic patients (2 women and 6 men) with rhegmatogenous retinal detachment (RRD). To the best of our knowledge, there are no reports on the measurement of the VEGF-B levels in vitreous humor of patients with ocular disease, making it difficult to use it as a reference value. Rhegmatogenous retinal detachment patients with no reports of other eye diseases or disorders were selected to serve as a control sample, minimizing the bias caused in the interpretation of the results. Only one eye from each patient was studied. The study enrolled 33 patients: 25 diabetic patients with PDR . These samples were collected at the beginning of the PPV (core vitrectomy). Tubes from the vitrectomy were disconnected and connected to a syringe (in coordination with vitrectomy aspiration at the beginning of the surgery). Before turning on the intravitreal infusion, an undiluted sample of vitreous was obtained by aspiration into a 2 mL syringe attached to the vitreous cutter. The sample was transferred to sterilized tubes, placed immediately on ice, and frozen at −80 °C until further analysis. Approximately 500 samples were collected from vitrectomized patients with several pathologies; of those, 33 samples were chosen for this study based on the following criteria: (1) patients with a confirmed diagnostic of DR (for the diabetic patient’s study arm), either with PDR or NPDR; (2) patients naïve to aflibercept treatment; and (3) patients with a sample volume that would allow the confirmation of the results through repeated enzyme-linked immunosorbent assay (ELISA) tests  for the determination of VEGF-B levels.3) from DR patients using SPECTRALIS OCT .Quantitative analysis of the optical coherence tomography (OCT) scans was performed to evaluate CRT (μm) and MV . Detection range was between 15.63–1000 pg/mL, and the sensitivity or the minimum detectable level was less than 5.5 pg/mL. The assay was conducted according to the protocol specified by the kit manufacturer. The enzymatic UV test (hexokinase method) was used for specific quantitative determination of glucose in vitreous using a Beckman Coulter analyzer AU2700  and Glucose reagent OSR6521. The results were reported in mmol/L.t-test and Mann–Whitney tests were used to analyze differences. Values of vitreous VEGF-B and glucose levels are reported as mean ± standard deviation . For all statistical analyses, a p-value ≤ 0.05 was considered to indicate statistically significant differences. Spearman’s ordinal correlation coefficient was used to analyze the relationship between quantitative variables because the variables had no normal distribution.Statistical analysis was performed with SPSS  version 20.0 for Windows . Student’s The study was approved by the Comissão de Ética para a Saúde (CES)/Ethics Committee for Health of the Centro Hospitalar de Leiria (Code: CHL-15481). All patients included in this study, which adhered to the tenets of the Declaration of Helsinki, gave their informed consent.The baseline characteristics of both groups are shown in p = 0.006).The mean VEGF-B concentration in vitreous humor was higher in the diabetic patients’ group (18.82 ± 1.44 pg/mL) in comparison with the control group of non-diabetic patients (17.90 ± 0.32 pg/mL) . This dip = 0.025) (Mean VEGF-B measurements were higher in vitreous humor of patients with PDR (19.03 ± 1.52 pg/mL) vs. NPDR (18.18 ± 0.96 pg/mL), and this difference was statistically significant (= 0.025) . p = 0.300) (Diabetic patients showed increased levels of glucose in vitreous humor (4.57 ± 2.07 mmol/L) when compared with non-diabetic patients (3.65 ± 2.39 mmol/L), but this difference was not statistically significant (= 0.300) .p = 0.032)  . sr = 0.441 (p ≤ 0.05), as seen in sr = 0.716 (p ≤ 0.01), as observed in The correlation between vitreous VEGF-B of DR patients and CRT was statistically significant, positive, and moderate: VEGF-B is a proangiogenic cytokine  that hasThe present study demonstrated that VEGF-B is significantly increased in diabetic patients with ocular disease in comparison with non-diabetic patients. This is an interesting finding as the VEGF-B levels have not been measured in the vitreous humor of human eyes. Moreover, the levels of VEGF-B were found increased in PDR in comparison to NPDR, and this difference was statistically significant. In addition, we found a positive correlation between VEGF-B vitreous levels and the CRT or MV measured by OCT, suggesting that overexpression of VEGF-B may influence these quantitative parameters in DR patients.It is uncertain whether the inhibition of VEGF-B is beneficial and improves patients’ visual acuity as well as structural outcomes, given the protecting and survival action of VEGF-B.Reports on the role of VEGF-B have provided controversial findings. Silvestre  demonstr186 in the nervous system in an animal study. Specifically, the neuroprotective effects of VEGF-B in combination with its very low angiogenic and permeability action. They reported its role as a neuroprotector, turning it into an appealing treatment for neurodegenerative diseases. Based on the findings of the above studies, it can be stated that VEGF-B plays a role in response to pathological conditions, and its upregulation may demonstrate benefits.VEGF-B therapy may lead to vessel survival and rescue of blood vessels from apoptosis; thus, it is being referred as a possible therapy in neurologic diseases, such as Parkinson’s disease. Confirming these findings, Poesen et al.  showed tHowever, other studies reported that VEGF-B plays a role as an inhibitor of choroidal and retinal neovascularization through VEGFR-1 and NRP-1. In his work, Zhang et al.  referredBased on the above studies describing the different functions of VEGF-B and the results of our investigation where VEGF-B appears to be overexpressed in DR in comparison with RRD, we suggest that VEGF-B targeted therapy by an anti-angiogenic drug may have a therapeutic effect on neovascular diseases. Due to the inhibition of VEGF-B, the treatment of vascular diseases of the retina may improve outcomes and have positive effects on the patients. It is important to acknowledge that VEGF-B does not act under normal physiological conditions, but instead in pathological conditions, which makes VEGF-B an attractive and safe therapeutic molecule.Lundquist O. and Österlin  demonstrVEGF-B was found significantly increased in DR patients, and this increase is significantly higher as the DR is at a more advanced stage. We suggest VEGF-B can offer an alternative and challenging therapeutic target in the treatment of neovascular conditions, such as diabetic eye disease. Randomized clinical trials are needed to be conducted in order to confirm the efficacy of anti-VEGF-B therapy. Furthermore, due to the cardioprotective and neuroprotective effect of this molecule, and taking into account that diabetic patients have multiple disease risk factors, studies targeting VEGF-B inhibition must be conducted carefully to determine its safety, particularly in these patients.


The aim of this study was to develop and assess a novel implantable, wireless-powered, light-emitting diode (LED) for near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT is a recently developed cancer therapy that uses NIR light and antibody-photosensitizer conjugates and is able to induce cancer-specific cell death. Due to limited light penetration depth it is currently unable to treat tumors in deep tissues. Use of implanted LED might potentially overcome this limitation.in vitro and to suppress tumor growth in implanted tumor-bearing mice.The wireless LED system was able to emit NIR light up to a distance of 20 cm from the transmitter coil by using low magnetic fields as compliant with limits for use in humans. Results indicated that the LED system was able to kill tumor cells in vivo. These results are encouraging as wireless LED systems such as the one here developed might be a possible solution to treat tumors in deep regions in humans. Further research in this area would be important.Results indicated that the proposed implantable wireless LED system was able to suppress tumor growth in vitro by using EGFR-expressing cells and HER2-expressing cells. The system was also tested in vivo in tumor-bearing mice.An implantable LED system was developed. It consisted of a LED capsule including two LED sources and a receiver coil coupled with an external coil and power source. Wireless power transmission was guaranteed by using electromagnetic induction. The system was tested  The trial went through phase I/II successfully, suggesting that NIR-PIT may be a promising cancer treatment.Near-infrared photoimmunotherapy (NIR-PIT) is a new type of cancer photo-therapy based on NIR light and antibody-photosensitizer conjugates . When thHowever, the procedure is not exempt from limitations. It is difficult to treat deep regions because of light absorption by tissues , 4. In pin vitro as well as in vivo in tumor bearing mice. To avoid potentially harmful implantation of batteries, power supply to the implanted LED was guaranteed by electromagnetic induction from an external transmitter coil to an implanted received coil coupled with the LED. The electromagnetic field was set to be compliant with standard limits for use in humans, i.e. lower than 0.1 mT.In this study, we developed a small implantable wireless LED for NIR light exposure and we have tested it As shown in Figure in vitro. The cytotoxic effect of NIR-PIT with the wireless LED was evaluated by using EGFR-expressing A431-GFP-luc cells. Severe cell damage such as cell swelling and bleb formation were induced by the combination of Pan-IR700 incubation and NIR light exposure  cells, and the severity of cell damage was dependent on the light, i.e. increased damage was observed with increasing light-dose. Fluorescence imaging showed that EthD-1 uptake increased with increasing light-dose, and that there was no cytotoxicity associated with Tra-IR700 alone nor with exposure to NIR light.Figure in vivo, we conducted experiments on tumor-bearing mice injected with A431-GFP-luc cells on both sides of the dorsum, as shown in Figure p = 0.066, 0.011, 0.010, respectively)  Figure . No sign) Figure . The bod) Figure .in vivo and reduce tumors in mice.Overall, these results suggested that the developed wireless LED was able to provide NIR-PIT Figure In this study, we have developed a prototype of implantable, wireless powered LED system for NIR-PIT. We used wireless power transmission through electromagnetic induction for supplying power from outside of the body to a receiver coil in the LED capsule, so to avoid implantation of batteries. Although the higher the electromagnetic field, the stronger the LED emission, the electromagnetic field in our system was limited to comply with the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guideline  so to avAnother important aspect for optimal development of implanted NIR-LED systems is related to reaching a trade-off between the light dose, which is desired to be high for increased treatment benefit, and the electromagnetic field, which needs to be low to avoid harmful effects on the human body. As a result, LED emission is limited due to limited electromagnetic field. Moreover, LED emission is also dependent on the distance between the LED/receiver coil and external transmitter coil and, also, between the small size of the LED capsule and receiver coil. Thus, especially in view of future applications in humans and in clinical settings, maximization of the LED emission is a key design issue. To try to partially tackle this issue, we reduced the error of resonant frequency between the transmitter and receiver coils and we improved the quality factor by using ferrite which has high magnetic permeability. The resulting LED emission was sufficient for treating tumors in mice.2) of the light source, but rather depends on the light dose in the tissue (J/cm2) [2 can be effective for NIR-PIT [in vivo experiments we had a maximum light dose of 320 J/cm2 which is above the minimal range and, as such, enabled tumor treatment. Another advantage of the proposed LED system compared to other solutions for NIR-PIT in deep tissues is related to the fact that the LED system is small and, once implanted, can irradiate tumors over a long period. On the other hand, conventional implantable light-delivering devices such as catheters cannot be left in the body for a long period and require recurrent implantation.The cytotoxic effects of NIR-PIT does not relate only to the power density (mW/cm (J/cm2) , 18. It  NIR-PIT , 19, 20.in vivo experiments tumors were exposed to NIR light continuously following implantation. During the treatment period, it is likely that the circulating mAb-IR700 from the first and subsequent injections accumulated in the tumor and contributed to more effective treatment. Use of NIR-PIT with wireless powered implanted LED might potentially be particularly useful to treat cancers in deep tissues in patients that have not responded successfully to conventional treatments. For example, cholangiocarcinoma or bile duct cancers mostly grow along with the biliary ducts and obstruct them, causing liver dysfunction and jaundice that might kill patients [It was proven that the therapeutic benefits of NIR-PIT can be enhanced by repeated drug administration and light exposure . The cirpatients , 24. To in vitro and in vivo.It is clear that further research is needed to fully understand the viability of the proposed device for use in humans as well as to investigate possible issues . Additionally, orthotopic tumor models growing in respective organs are surely superior to the subcutaneously xenografted tumor model used in this study for evaluating clinical relevance of this LED system –27. HoweFigure The LED capsule was about 7 mm in length and was composed of a receiver coil, a capacitor and two LEDs as shown in Figure ®), a fully humanized IgG2 monoclonal antibody (mAb) directed against the epidermal growth factor receptor (EGFR), was purchased from Takeda Pharmaceutical Co. Ltd. . Trastuzumab (Herceptin®), 95% humanized IgG1 mAb directed against the human EGFR type-2 (HER2), was purchased from Chugai Pharmaceutical Co., Ltd. . Water-soluble silicon-phthalocyanine derivative, IRDye700DX NHS ester (IR700) was obtained from Li-COR Bioscience . All the other chemicals used were of reagent grade.Panitumumab (Vectibix2HPO4 (pH 8.5) at room temperature for 2 h. The mixture was purified with a gel-filtration column , and derived Panitumumab-IR700 (Pan-IR700) or Trastuzumab-IR700 (Tra-IR700). Protein concentration was determined with Modified Lowry Protein Assay Kit  by measuring the absorption at 750 nm with a microplate reader . The concentration of IR700 was measured by absorption at 689 nm with a spectrophotometer  to confirm the number of fluorophore molecules to each mAb. The number of IR700 per mAb was approximately equal to three.Panitumumab or Trastuzumab  was incubated with IR700  in 0.1 M NaGreen fluorescence protein (GFP), luciferase-expressing A431 (A431-GFP-luc) cells, and HER2-gene-transfected NIH3T3 (3T3-HER2) cells were prepared by Dr. Kobayashi's laboratory. Cell lines were grown in RPMI-1640  containing 10% fetal bovine serum  and 1% penicillin/streptomycin  in tissue culture dishes in a humidified incubator at 37° C in an atmosphere composed of air (95%) and carbon dioxide (5%).2). Power density was measured with an optical power meter . The cells were observed by fluorescence microscope  2 h after the onset of light exposure. To detect EthD-1, a combination of 480–550 nm excitation filter and 590 nm long pass emission filter was used. To detect IR700, a combination of 673–748 nm excitation filter and 765–855 nm band pass emission filter was used.A431-GFP-luc cells or 3T3-HER2 cells were seeded on 35 mm culture dishes and incubated for 24 h. After washing with phosphate buffered saline (PBS), phenol-red-free RPMI-1640 supplemented with 15 mM HEPES was added. Pan-IR700 or Tra-IR700 was then added to the culture medium at 5 μg/mL and incubated for 1 h at 37° C. Ethidium homodimer-1  was used to detect dead cells. The cells were irradiated from the bottom of the dish with NIR light using the wireless LED for 0, 3, 10, 30, and 60 min , phenol-red-free RPMI-1640 containing 150 μg/mL D-luciferin was administered to A431-GFP-luc cells. It was analyzed by using a luminescent imaging analyzer LAS-4000 mini . Image analysis was conducted with ImageJ software (http://rsb.info.nih.gov/ij/).For BLI, after irradiation from the bottom with NIR light using the wireless LED for 1, 3, 5, 10, 20, and 30 min  were purchased from Japan SLC, Inc. . During procedures, mice were anesthetized with isoflurane.2 × 0.5. Body weight was also measured. IR700 fluorescence was evaluated using the FluorVivo Imaging System  . The ClearView Imaging System  was used to obtain fluorescence images of GFP . The region of interest was manually determined on each tumor area. Fluorescence image analysis was conducted with ImageJ software.Five million A431-GFP-luc cells in PBS were injected subcutaneously in right and left sides of the dorsum, and the experiments were conducted 6 days following cell injection. The greatest longitudinal diameter (length) and the greatest transversal diameter (width) of tumors were measured by using a caliper. Tumor volume was calculated following : volume Mice were given intravenous injection of 100 μg of Pan-IR700 on day 0 and day 3. The mice had two tumors on the dorsum and were randomized into two groups: (1) treated mice: a tumor with NIR-LED (LED (+)_NIR (+)) and a tumor without LED (LED (−)_NIR (−)); (2) non-treated mice: a tumor with implanted dummy LED (not emitting NIR light) (LED (+)_NIR (−)) and a tumor without LED (LED (−)_NIR (−)) Figure . The dumTo evaluate the histological changes, mice were euthanized on day 6. Extracted tumors were frozen in optimal cutting temperature compound, and 10 μm frozen slice sections were prepared. Hematoxylin and eosin (H&E) staining was performed according to standard protocols. The slides were imaged with an Olympus BX41 microscope  with 4× and 20× objective lenses.t tests. Values of p < 0.05 were considered statistically significant.The measured quantities were expressed as means ± SEM (standard error of the mean). Statistical analyses were carried out using JMP Pro 12.2.0 software . Possible differences in mean values were assessed by using student's


Histone deacetylase 3 (HDAC3) has been recently identified as a potential target for the treatment of cancer and other diseases, such as chronic inflammation, neurodegenerative diseases, and diabetes. Virtual screening (VS) is currently a routine technique for hit identification, but its success depends on rational development of VS strategies. To facilitate this process, we applied our previously released benchmarking dataset, i.e., MUBD-HDAC3 to the evaluation of structure-based VS (SBVS) and ligand-based VS (LBVS) combinatorial approaches. We have identified FRED (Chemgauss4) docking against a structural model of HDAC3, i.e., SAHA-3 generated by a computationally inexpensive “flexible docking”, as the best SBVS approach and a common feature pharmacophore model, i.e., Hypo1 generated by Catalyst/HipHop as the optimal model for LBVS. We then developed a pipeline that was composed of Hypo1, FRED (Chemgauss4), and SAHA-3 sequentially, and demonstrated that it was superior to other combinations in terms of ligand enrichment. In summary, we present the first highly-validated, rationally-designed VS strategy specific to HDAC3 inhibitor discovery. The constructed pipeline is publicly accessible for the scientific community to identify novel HDAC3 inhibitors in a time-efficient and cost-effective way. Histone deacetylases (HDACs) are a family of enzymes that catalyze histone deacetylation, a major form of posttranslational modifications in mammals. HDACs work together with their counterpart, i.e., histone acetyltransfereases (HATs), to maintain acetylation homeostasis and regulate gene transcription ,2,3,4. FIn the whole family of HDACs, HDAC3 has gained more and more attention in recent years and showing its potential as a target for modern drug discovery. HDAC3 maintains the primarily deacetylated state of nuclear factor κB (NF-κB), which is a positive regulator of inflammatory gene expression . Due to In recent years, the relationship between HDAC3 and learning/memory has been gradually uncovered. Wood’s group identified HDAC3 as a negative regulator of long-term memory formation, and demonstrated HDAC3 inhibition was able to improve cognitive impairments . They alHdac3 alone or by using the HDAC1/2/3-selective inhibitor, MS-275 , are expressed in the pancreatic β-cells . The inh-275 cf.  19]. Th. ThHdac3-275 cf.  and HDACCurrently, many ligands are able to inhibit HDAC3. However, most of them belong to anti-cancer pan-HDAC inhibitors and rarely show specific inhibition for HDAC3. To the best of our knowledge, RGFP966 and BRD3308 are the only HDAC3-specific inhibitors that show therapeutic effects on diseases other than cancers. Therefore, it remains an emerging area to discover novel HDAC3Is for the treatment of those diseases. We have been working on identification of novel-scaffold HDAC inhibitors by using computer-aided drug design (CADD) and cheminformatics ,23. MostWe explored the potentials of 10 scoring functions implemented in DS 2016  to improve ligand enrichment. As FRED performed the best among all programs, all docking poses from it became the input for the “score ligand poses” module in DS. Unfortunately, this rescoring approach did not further improve ligand enrichment of FRED (Chemgauss4), as all ROCE 0.5%, ROCE 1%, and ROC AUC values of those explored scoring functions were lower than those of FRED (Chemgauss4) cf. .In addition to docking programs and scoring functions, we hypothesized that ligand-induced-fit protein models may enrich HDAC3Is better that the apo structure . To test this hypothesis, we generated models by ligand-induced-fit docking of five HDAC3Is cf.  into theThe total number of ligand-induced-fit protein models was 42. As FRED (Chemgauss4) was validated as the best docking/scoring program, it was used to dock and score all actives and decoys in MUBD-HDAC3 against every ligand-induced-fit model. Ligand enrichments of these models along with the use of FRED (Chemgauss4) are shown in We further analyzed the ligand-induced-fit protein models from the same ligand and selected the best model, i.e., model of highest enrichment for each ligand. As shown in The above data confirmed that inclusion of ligand-induced-fit effect by “flexible docking” in DS was able to generate protein-ligand complexes that (1) contained essential interactions with a zinc ion; and (2) more importantly, possessed better ligand enrichment than the apo structure. In addition, the use of a benchmarking calculation based on MUBD-HDAC3 was effective in selecting the optimal structural model.Since five HDAC3Is in the ligand set for molecular modeling were observed to share common features in different protein-ligand complexes, we used Catalyst/HipHop to generate common feature pharmacophore models. According to Ligand enrichment is a preferred metric for predicting model performance in real-world screening. The evaluation outcome based on MUBD-HDAC3 is listed in Interestingly, we observed the inconsistency between rank and enrichment cf.  for a feIn addition to common features, five HDAC3Is in the ligand set for molecular modeling had their own features in terms of both chemical structure and binding pose. ROCS program was applied to generate models to uncover these differences. As for ROC AUC, the values for all pairs of model/metric are all around 0.50 and their pairwise difference is within 0.10. This indicated in general that ligand enrichment of a shape-based similarity search was no better than in random enrichment. However, all pairs of models/metrics, except for SAHA-3L/ST , performed better in early recognition than random selection. The best metric varies for each model. The best metric for 15k-5L was TC, a combined score of ST and CT. With the use of CT, 8d-7L showed best enrichment. The metrics that result in optimal enrichments for R306465-7L, SAHA-3L and MS-275-4L are CT, TC, and CS (combined score of ST and SC), respectively. Among them, the best pair was SAHA-3L/TC, whose early enrichments in terms of ROCE 0.5% and ROCE 1% were 20.60 and 17.96. Nevertheless, it was still not as good as Hypo1 from Catalyst/HipHop .Via exhaustive evaluation, we have identified (1) the ligand-induced-fit protein model, i.e., SAHA-3 combined with FRED (Chemgauss4) led to best ligand enrichment, in particular early recognition; (2) the common feature pharmacophore model, i.e., Hypo1 generated by Catalyst/HipHop was superior to shape-based models generated by ROCS; and (3) The optimal SBVS approach performed better than pharmacophore filtering, i.e., Hypo1. Based on the outcome, we developed a pipeline to rapidly and effectively screen large-scale chemical libraries. This pipeline was composed of pharmacophore filtering by Hypo1, followed by use of FRED docking against SAHA-3. Notably, this combination showed better performance than the use of Hypo1 alone. Meanwhile, it did not significantly impair the good early recognition of molecular docking alone, as its ROCE 0.5% and ROCE 1% values remained close, i.e., 72.10 and 38.49 cf. . In lighIn addition, we evaluated three other combined pipelines, i.e., Hypo1_GOLD_apo, Hypo1_LibDock_apo, and Hypo1_FRED_apo. These pipelines were the most probably constructed and applied ones for HDAC3Is discovery, if ligand enrichment assessment  was not performed. The reasons were: (1) Hypo1 had a high rank score in Catalyst/HipHop and, thus, would undoubtedly become the first component of the discovery pipelines cf. ; (2) anyFive potent and diverse HDAC3Is were selected from the literature, i.e., 15k , 8d 30]30], R306http://www.xswlab.org/, accessed in January 2016) [As mentioned before, MUBD-HDACs can be applied to benchmark both LBVS and SBVS approaches in an unbiased manner . Therefory 2016) . It inclBased on a list of ranked scores of ligands and decoys in MUBD-HDAC3, a receiver operating characteristic (ROC) curve was generated to evaluate ligand enrichment of different approaches. A ROC curve is a plot that describes the true positive rate against the false positive rate at various discrimination threshold settings. It is commonly used in data analysis to evaluate the performance of a binary classifier in discriminating the positive (active) from the negative (decoy). The more specific metrics from the ROC curve were ROC area under the curve (ROC AUC) and ROC enrichment (ROCE) values. The former represented overall enrichment, while the latter measured early recognition. Since early recognition has more practical significance than overall enrichment in real-world screening campaigns, ROCE 1% and ROCE 0.5% were given priority for determining the optimal approach.  The ROCEThree docking programs that we had access to, LibDock implemented in DS, GOLD , and FRED  were applied to screen MUBD-HDAC3. Each program had its inherent scoring function, i.e., Libscore for LibDock, Chemscore for GOLD, and Chemgauss4 for FRED. The ready-to-use apo structure from MUBD-HDAC3 was directly used as a receptor for molecular docking. In molecular docking simulations using the three programs, zinc ion in the binding site was treated as rigid.For LibDock, the binding site was defined as a sphere centered on zinc ion in the catalytic site with a radius of 12 Å. All compounds in MUBD-HDAC3 were converted to 3D conformers by the “FAST” method in DS, with 255 as the maximum conformations and 20 kcal/mol as an energy threshold. All conformers were docked against the binding site and scored by Libscore. A maximum of 30 poses were saved for each compound. For GOLD, it was not necessary to generate a multi-conformer database before molecular docking. This program used the initial low-energy conformation of each compound as an input, and applied a genetic algorithm to optimize the pose in the binding site. The binding site was defined as exactly the same as that for LibDock. The zinc ion was retained and predefined to form tetrahedral coordination geometries during molecular docking. The docking mode was set as “7–8 times speed-up” and each compound was docked 30 times. All poses were scored by ChemScore. For molecular docking by FRED, firstly OMEGA   is a shape-based superposition method and well known for its accuracy and speed ,40. BaseDue to low computational cost, LBVS approaches are usually set up ahead of SBVS approaches in a pipeline for large-scale VS. Accordingly, the best model obtained from the evaluation of LBVS approaches and the optimal SBVS approaches, e.g., docking program/scoring function and the ligand-induce-fit protein model, were integrated into a pipeline. This pipeline was then benchmarked based on the MUBD-HDAC3 dataset. To do this, the compounds in MUBD-HDAC3 were filtered by the best pharmacophore/shape-based model. Those compounds that passed the filter were further docked against the optimal structural model of HDAC3 by the selected docking program and scored by the best scoring function. For the purpose of comparison, those compounds that passed the filter were also docked against the apo structure using a common docking/scoring program. The outcomes from these combined LBVS and SBVS approaches were then analyzed.In this paper, we used MUBD-HDAC3 to unbiasedly validate the screening power of various SBVS and LBVS approaches and select the optimal ones. Stepwise evaluation of docking programs and scoring functions identified FRED (Chemgauss4) as optimal for enriching HDAC3Is based on the apo structure. Docking against multiple ligand-induced-fit protein models by FRED (Chemgauss4) further uncovered SAHA-3 as a structural model of better screening power. Comparative analysis of common feature pharmacophore models by Catalyst/HipHop and shape-based models by ROCS demonstrated Hypo1 from the former performed the best in terms of early enrichment. Based on the outcome, we presented a versatile pipeline, i.e., Hypo1_FRED_SAHA-3, to rapidly and effectively enrich HDAC3-targeted active compounds from large-scale chemical libraries. We have demonstrated this rationally developed pipeline was superior to other combinations in terms of ligand enrichment, and this highlighted the importance of benchmarking calculations in decision-making.https://www.dropbox.com/sh/5w9d6y8m77hk9pb/AAA2QoItV7wEeFS4p1ZyQzMda?dl=0). Our immediate goal is to apply this pipeline to screen chemical libraries from different sources and test potential hits for their HDAC potency/selectivity profile as well as their anti-diabetic effect.To the best of our knowledge, this work is the first to attempt to develop VS pipelines for intentionally screening HDAC3-specific inhibitors. The models associated with this VS pipeline, i.e., Hypo1 and SAHA-3, and their usages are publicly accessible via the following link (available on:


The aim of our study is to assess the frequency of detection of PET-positive computed tomography (CT)-negative skeletal metastases (SM) and determine the impact of such detection on staging and/or management in patients who had FDG PET/CT as part of the cancer work-up.We retrospectively reviewed 2000 18F-FDG PET/CT scans of known cancer patients. A log was kept to record cases of suspected SM with or without bone changes from the low-dose non-contrast CT. The presence or absence of SM was evaluated based on available pathological and clinical data. The impact of detection of such lesions on cancer staging and/or management was evaluated by a board certified oncologist.Of the 2000 cases, 18F-FDG PET/CT suggested SM in 146/2000 (7.3%). Of those 146 cases, 105 (72%) were positive on both PET and CT. The remaining 41 (28%) had PET-positive CT-negative bone lesions. SM was confirmed in 36/41 (88%) PET-positive/CT-negative cases. This was based on biopsy, imaging, or clinical follow-up. The detection of PET-positive CT-negative SM did not change staging or management in 7/36 (19.4%). However, staging and/or management was affected in 29/36 (80.6%).SM is not uncommon in 18F-FDG PET/CT, as it accounts for 146/2000 (7.3%) of cases. PET demonstrated FDG-avid SM without a CT abnormality in at least 36/146 (25%). Patients staging and/or management changed in 29/36 (80.5%). We concluded that 18F-FDG PET is sensitive in the detection of SM with significant impact on staging and/or management. Early diagnosis of cancer remains to be of paramount importance to maximize a patient’s long-term survival and reduce various neurological, hematological, and orthopedic complications that may arise. Skeletal metastasis (SM) is a frequently encountered and important complication of cancer which can lead to intolerable pain. Bone metastases are strongly associated with increased risk of death and increased risk of skeletal-related events (SREs) , 2. SREsAfter obtaining Saint Louis University Institutional Review Board (IRB) approval, we retrospectively reviewed 2000 consecutive 18F-FDG PET/CT scans of known cancer patients. The vast majority of patients had top of the head to bottom of the feet 18F-FDG PET/CT, as it is the standard of care in our institution. A log was kept for cases with suspected SM. PET-positive cases were further evaluated for the presence of bone window changes in the CT. The presence or absence of SM in PET-positive/CT-negative cases was determined based on all available pathological and clinical data. The impact of detection of SM on cancer staging and/or management was evaluated by a board certified oncologist.18F-FDG PET/CT scans were acquired using PET/CT scanner  with an axial co-scan range of 193 cm. Per institutional protocol, all patients were instructed to fast at least 4 h prior to receiving the radiopharmaceutical injection. Blood glucose level was <200 mg/dl in all patients. On the day of the exam, intravenous injection of 5.18 MBq/kg (0.14 mCi/kg) of F18-FDG was administrated. For the uptake phase, patients sat in a quiet room without talking for about 60 min.The CT component of the PET/CT scanner has 64 multidetector helical CT with a gantry port of 70 cm. The parameters of CT detectors were set as follow for 20–21 bed acquisitions: 120–140 kV and 33–100 mAs (based on body mass index), 0.5 s per CT rotation, pitch of 0.9, and 512 × 512 matrix data were used for image fusion and the generation of the CT transmission map. The CT images were obtained without oral or IV contrast administration according to the standard PET/CT protocol at our institution.The PET component of the PET/CT scanner is composed of lutetium–yttrium oxyorthosilicate (LYSO)-based crystal. Emission scans were acquired at 1–2 min per bed position. The FOV was from the top-of-head to the bottom-of-feet in vast majority of patients. The three-dimensional (3D) whole-body WB acquisition parameters were 128 × 128 matrix and 18 cm FOV with a 50% overlap. Processing used the 3D Row Action Maximum Likelihood Algorithm (RAMLA) method. Total scan time per patient was approximately 20–45 min.PET/CT images were retrospectively evaluated on the Gemini TF extended brilliance workstation (EBW) by a board certified nuclear medicine physician and a fellowship trained radiologist. Cases were categorized into two groups: the first group was cases with PET-positive and CT-positive bone lesions, in which the CT of the PET/CT demonstrated morphological changes whether lytic, sclerotic, or mixed. The second group was PET-positive and CT-negative for bone lesions, in which the CT of the PET/CT failed to demonstrate definite morphological bone or bone marrow changes. No minimum size, number, or standardized uptake value criterion was assigned to the SM for inclusion into the study. A thorough review of the medical records, including clinical notes, pathology reports, and radiology reports, was performed by a board certified oncologist to confirm the diagnosis of SM and convey the impact of its detection on cancer staging and/or management.n = 8), lymphoma (n = 7), lung cancer (n = 6), melanoma (n = 4), leukemia (n = 3), multiple myeloma (n = 2), gastrointestinal cancers (n = 2), and others (n = 4). Of the remaining 5/41 (12%), SM was not confirmed since one patient had a negative bone biopsy and the other four patients had no follow-up. The impact of detection of PET-positive CT-negative SM on staging and/or management was determined by a board certified oncologist. The detection of PET-positive/CT-negative SM did not change staging or management in 7/36 (19.4%), because these patients had widespread disease. In total, staging and/or management was affected in 29/36 (80.6%). This is further divided into patients with a change in staging and management in 4/36 (11%), change in staging only in 1/36 (3%), and change in management only in 24/36 (67%) . In those 146 cases, 105 (72%) were positive on both PET and CT. The remaining 41 (28%) had PET-positive/CT-negative bone lesions   of cases. We related the discrepancy between PET and CT in the detection of SM to the mechanism by which FDG is accumulated into the SM by viable and metabolically active tumor cells, which is visible by PET before the detection of anatomic changes in the CT. Detection of such lesions changed staging and/or management in 29/36 (80.6%) of patients who needed special treatment to SM. Although we did not evaluate the effect of the detection of SM on overall survival (OS), research studies found that detection of SM may have an impact on prognosis and OS in some cancers. This is particularly true in breast cancer patients with bone marrow metastases and is even worse if it is complicated by pathological fracture . Demir e% of case< 0.001) –29.Our study is not without limitations. We compared the sensitivity of PET to the low-dose CT of the PET/CT because it is the standard of care in our institution. Indeed, most of the patients did not have full dose diagnostic CT available to compare to the PET/CT. However, it is not likely that the diagnostic accuracy of contrast CT would have been significantly better than on low-dose non-contrast CT. Two of the patients had full dose diagnostic CT done within a month from the 18F-FDG PET/CT and neither demonstrated corresponding changes in CT bone window Figures  and 5. AIn our study, SM was not uncommon in 18F-FDG PET/CT, as it accounts for 146/2000 (7.3%) of our cases. PET demonstrated FDG-avid SM without a CT abnormality in at least 36/146 (25%) which resulted in a staging and/or management change in 29/36 (80.5%). We concluded that 18F-FDG PET may be sensitive in the detection of SM with a significant impact on patient staging and/or management.FA contributed in data gathering and writing of the manuscript. RM and MO contributed in image analysis and writing of the manuscript. HF and YT contributed in writing of the manuscript and detection of the impact of PET-positive CT-negative SM on management and/or staging. BA and MO reviewed the manuscript and edited the discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Insufficient response to antipsychotics constitutes a challenge in the treatment of patients suffering from schizophrenia. Treatment resistances have been linked to a normal striatal dopamine system. We aim to stratify antipsychotic-naïve first-episode patients based on striatal dopamine synthesis capacity (DSC) measured with positron emission tomography (PET). We hypothesize that patients who respond to treatment have an increased DSC at baseline compared to non-responders and healthy controls (HC).The current data have been collected as a part of a multimodal first episode study. Patients are examined before and after 6 weeks treatment with flexible doses of Aripiprazole.PET: Dynamic scans are performed in an integrated PET-CT scanner using the tracer 3,4-dihydroxy-6-[18F]fluoro-L-phenylalanine (18F-FDOPA). Duration of scanning is two times one hour, with half an hour break.Arterial blood samples are collected during the scanning sessions and provide information on the ratio between intact 18F-FDOPA and the primary metabolite. The input functions used for this data analysis are image derived based on the largest cerebral vessels and metabolite corrected.Regions of interest (ROIs) are manually drawn around the cerebellum and semi automatically around the basal ganglia. In this preliminary work, DSC values are based on Ki parameters obtained from slopes on Patlak plots.DSC has been measured at baseline on 16 patients  and 18 HC , with no significant difference in age or gender between groups.At baseline patients had a PANSS total score of 74 (SD 9.6) and GAF total score of 35 (SD 5.2).No significant difference in Ki at baseline was shown between patients and HC. Clinical follow-up data was available on 12 patients. They received a mean Aripiprazole-dose of 9.3 (SD 3.9). Paired t-test at showed a significant effect of treatment with a follow up PANSS total of 55  and GAF total of 49 .Six patients were characterized as responders, and six as non-responders using the Nancy Andreasen remission criteria. There were no baseline differences in PANSS or GAF scores between responders and non-responders. Nor did the dose of Aripiprazole differ between these groups. There was however significant difference in the GAF total score at follow up (p =0.001), as GAF was 59 (SD 9.3) for responders and 39 (SD 5.3) for non-responders.Mean Ki-values at baseline was 0.79 (SD 0.2) for non-responders, 0.88 (SD 0.2) for responders and 0.91 (SD 0.2) for HC. One way ANOVA showed no significant group difference.Although not significant, we found a slightly lower Ki-value at baseline for non-responders compared to baseline Ki-values for responders and HC in these preliminary analyses. This was unexpected, but should be taken with precaution, as the results represent work in progress.Inclusion of subjects and data-analyses is ongoing, and data analysis will be more extensive in spring 2018, especially regarding the methodology:The current image derived input function suffers from partial volume effects (PVE). To account for PVE and other factors the image data will be co-registered with T1-weighted MRI data and normalized to standard space in order to use a standard anatomical atlas to help define the ROIs. Finally, as mentioned earlier, arterial samples are collected and we plan to use arterial input functions to correct for the complicated kinetics of the tracer. To improve the Ki estimation we will use the metabolite corrected arterial plasma curve as input function and compare these results with the current method.


In neurodegenerative disorders, such as Parkinson's disease (PD), alpha-synuclein (α-syn) accumulates to induce cell death and/or form a cytoplasmic inclusion called Lewy body (LB). This α-syn-related pathology is termed synucleinopathy. It remains unclear how α-syn accumulation expands during the progress of synucleinopathy in the human brain. In our study, we investigated the patterns of distribution and propagation of forebrain neurons expressing α-syn in aged macaques. It was found that the occurrence of α-syn-positive neurons proceeded topologically based on the midbrain dopamine pathways arising from the substantia nigra and the ventral tegmental area where they were primarily observed. In the nigrostriatal or mesolimbic dopamine pathway, the age-dependent increase in α-syn-positive neurons was evident in the striatum or the nucleus accumbens, respectively. Concerning the nigrostriatal pathway, a mediolateral or rostrocaudal gradient was seen in the substantia nigra or the striatum, respectively, and a compensatory increase in dopamine transporter occurred in the striatum regardless of the decreased dopamine level. In the mesocortical dopamine pathway, α-syn-positive neurons appeared in the prefrontal and then motor areas of the frontal lobe. Given that neither LB formation nor clinical phenotype manifestation was detected in any of the monkeys examined in the present study, aged macaques may be useful as a potential presymptomatic model for PD and LB-related neuropsychiatric disorders. Alpha-synuclein (α-syn) is a 140-amino acid protein that is localized specifically to the presynaptic terminals and nuclei of neuronal cells, though its physiological functions remain to be known . In at lClinical reports have shown that dementia is developed or accompanied in many of PD patients to induce Parkinson’s disease with dementia (PDD) or dementia with Lewy bodies (DLB), respectively ,14. LikeTo understand the pathophysiology of synucleinopathy, it is important to know how α-syn accumulation expands in the process of normal aging. However, the following problems make it very difficult to analyze this issue using human brains. First, the human brain is too huge to undergo pathological examinations throughout the brain. Second, it is quite painful to collect brain samples at individual ages with no history of neuropsychiatric disorders. Instead of human brains, the application of nonhuman primate brains is meritorious for exploring the extent of α-syn accumulation during normal aging. In fact, Chu et al.  performeIn the present study, we investigated the patterns of distribution and propagation of α-syn-positive neurons in aged monkey brains, in order to elucidate when and where synucleinopathy starts and how synucleinopathy develops. Particular attention was paid to forebrain structures, including the frontal cortical areas. In addition, closer analysis was carried out to clarify the age-dependent progress in the α-syn pathology of the nigrostriatal dopamine system. For this work, we used a total of 14 macaque monkeys without abnormal movement sign or past disease history. Their ages ranged 10–31 years old. According to previous reports ,25, the The experimental protocol used for this study including animal sacrifice was approved by the Animal Welfare and Animal Care Committee of the Primate Research Institute, Kyoto University (Permission Number: 2013–008). All experiments were conducted in accordance with the Guidelines for Care and Use of Nonhuman Primates  issued by the institute.Macaca fuscata), six cynomolgus monkeys (Macaca fascicularis), and one rhesus monkey (Macaca mulatta)] of either sex weighing 4.0–8.5 kg were used for this study. As described in Fourteen macaque monkeys [seven Japanese monkeys  at a 1:2,000 dilution, mouse monoclonal antibody against phosphorylated α-syn  at a 1:5,000 dilution, rabbit polyclonal antibody against TH  at a 1:1,000 dilution, or rat monoclonal antibody against DAT (Millipore) at a 1:1,000 dilution, in PBS containing 0.1% Triton X-100 and 2% normal donkey serum. After 48 h, the sections were washed in PBS and incubated in the same fresh medium containing biotinylated goat anti-mouse/anti-rabbit/anti-rat IgG antibody  for 2 h at room temperature, followed by avidin-biotin-peroxidase complex  for 2 h at room temperature. Subsequently, the sections were reacted in in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.04% diaminobenzidine tetrahydrochloride , 0.04% NiCl2, and 0.002% H2O2. Finally, the sections were mounted onto gelatin-coated glass slides, air-dried, counterstained with 1% Neutral red, and coverslipped. Another series of sections was Nissl-stained with 1% Cresyl violet.Following sedation with ketamine hydrochloride  and xylazine hydrochloride , the monkeys were deeply anesthetized with an overdose of sodium pentobarbital  for perfusion-fixation. The monkeys were transcardially perfused with 0.1 M phosphate-buffered saline , followed by 10% formalin in 0.1 M phosphate buffer . The brains were removed from the skull, postfixed in the same fresh fixative overnight, and saturated with 30% sucrose in PB at 4°C. Frontal sections were then cut serially at 50 μm thickness on a freezing microtome and processed for immunohistochemical staining with the avidin-biotin peroxidase complex (ABC) method. A series of every ten sections were immunostained for α-syn, phosphorylated α-syn, tyrosine hydroxylase (TH), or dopamine transporter (DAT). Especially for TH and DAT immunostaining, the sections through the striatum and the substantia nigra were used. After washes in PBS, the sections were soaked in 0.3% HFor double immunofluorescence staining for α-syn and TH or TH and NeuN, the sections through the substantia nigra were incubated with a cocktail of the mouse anti-α-syn antibody  and the rabbit anti-TH antibody , or a cocktail of the rabbit anti-TH antibody  and mouse monoclonal antibody against NeuN . The sections were then incubated with a cocktail of Alexa Fluor 555-conjugated donkey anti-mouse IgG antibody  and Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody . Images of sections were digitally captured at x4 or x10 magnification using an optical microscope equipped with a high-grade charge-coupled device (CCD) camera .Anatomical structures were identified by referring to “A Combined MRI and Histology Atlas of the Rhesus Monkey Brain in Stereotaxic Coordinates”  and “The2. The entire process was done on digitally captured x40 images by the aid of Adobe Photoshop CS5 software and Adobe Illustrator CS5 software. When the numbers of α-syn-positive and TH-positive neurons were compared in the medial vs. lateral parts of the SNc, 3 regions of interest (ROIs) each of which was 200 μm x 200 μm were placed in both parts along the mediolateral axis. In each part, the cell counts obtained in the ROIs were averaged and converted into the number in the area of 1 mm2. The same process as described above was taken for analysis.In individual monkeys, the numbers of α-syn-positive and TH-positive neurons were counted in the ventral midbrain, including the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). Cell counts were performed in every ten sections 50 μm thick  on each side, and the data obtained were averaged and expressed as the number in the area of 1 mmThe number of α-syn-positive neurons was also counted in forebrain structures, including the frontal cortical areas. In each structure, cell counts were performed in every ten sections 50 μm thick  on each side. On each section, 3 ROIs of 200 μm x 200 μm were placed. The data obtained in the ROIs were averaged, and then the neuron number per section was calculated.For double immunofluorescence staining for α-syn and TH or TH and NeuN, cell counts were carried out in 3 ROIs of 200 μm x 200 μm that were placed within nigral zones where clusters of TH-positive neurons were located in every ten sections .In individual monkeys, the density of TH immunoreactivity or DAT immunoreactivity was measured in the striatum. Measurements of optical density (OD) were performed in 2 ROIs of 500 μm x 500 μm placed in each of the caudate nucleus and the putamen in every ten sections  on each side. The OD was measured by the aid of Adobe Photoshop CS5 software and Adobe Illustrator CS5 software, and the value obtained in the ROIs in each section was averaged and expressed as a ratio to a control value . The entire process was done on digitally captured x40 images by the aid of Adobe Photoshop CS5 software. Finally, the mean OD ratio per section was calculated. When the OD ratio was compared in the rostral vs. caudal levels of the striatum, the boundary was set at the level of the anterior commissure.The cell counts and OD ratios were analyzed by Mann-Whitney test. Correlations between the cell counts and the age, or those between the OD ratios and the age were examined. Significance for each analysis was set a priori at 0.05. All the statistical analyses were done by the aid of IBM SPSS Statistics 22 package and Microsoft Excel 2010.As listed in p = 0.0362, Figs p = 0.0278, Figs In the youngest 10-year-old monkey, α-syn-positive neurons were found prominently in the SNc . Therefop = 0.0276, putamen; p = 0.0045, Figs p = 0.0354, The density of TH-immunoreactive fibers and terminals in the striatum was gradually reduced as the age went , we examined whether or not phosphorylated α-syn-positive neurons occur in the aged brain. In all of these monkeys, only a few faintly-immunostained neurons were observed in the SNc and the amygdala . No otheIn present study, we analyzed the patterns of distribution and propagation of forebrain neurons expressing α-syn in aged macaques. The main findings were as follows: (1) α-Syn-positive neurons appeared topologically along the midbrain dopamine pathways originating in the substantia nigra and the ventral tegmental area where they were primarily located. (2) Regarding the nigrostriatal or mesolimbic dopamine pathway, α-syn-positive neurons were increasingly observed in the striatum or the nucleus accumbens, respectively, in an age-dependent fashion. (3) In the nigrostriatal pathway, a mediolateral or rostrocaudal gradient was seen in the substantia nigra or the striatum, respectively. (4) Concerning the mesocortical dopamine pathway, α-syn-positive neurons were found in the prefrontal and then motor areas of the frontal lobe.It is generally accepted that in neuropathological conditions, phosphorylated α-syn abnormally accumulates within the cytoplasm and aggregates to form insoluble fibrils characterized by the intracellular inclusion LB . This α-According to the hypothesis proposed by Braak , in PD pIt has been shown that there is topological arrangement in the nigrostriatal dopamine pathway –39. NeurContrary to the decreased density of TH immunoreactivity, DAT immunoreactivity in the striatum remains unchanged even in the far-aged brains. Striatal DAT is localized at the presynaptic terminals of nigral dopamine neurons to promote reuptake of dopamine that is released from the terminals . This clIt has recently been revealed that the α-syn pathology related to disease development is transmitted based on the neural linkage –23. The Our results have further demonstrated that the number of α-syn-positive neurons is increased in the VTA as the age goes, though they appear less rapidly and drastically than in the SNc. In conjunction with the medial SNc, the VTA gives rise to the mesolimbic and mesocortical dopamine pathways ,49. The Moreover, neurons in the VTA/medial SNc provide the mesocortical dopamine pathway to the frontal lobe ,38,52, aThe present results indicate that the α-syn pathology expands primarily along the dopaminergic neuron connectivity in a topological fashion. At the early stage of aging, α-syn-positive neurons emerge in midbrain dopamine neurons. From the SNc, the occurrence of α-syn-positive neurons proceeds to the striatum through the nigrostriatal dopamine pathway, while from the VTA/medial SNc, it extends to the limbic structures via the mesolimbic dopamine pathway, and to the frontal cortical areas via the mesocortical dopamine pathway. This propagation pattern of α-syn-positive neurons seems basically compatible to the human LB pathology. In none of the aged macaque monkeys tested in the present study, however, either LB formation or clinical manifestation (at least PD-like motor signs) was detected. This indicates that the α-syn pathology is not necessarily disease-specific, but is common to normal aging. It should also be noted here that not only the propagation associated with the midbrain dopamine systems, but also the dementia-related changes probably underlie the α-syn pathology, especially in the amygdala, the hippocampus, the anterior cingulate cortex, and the orbitofrontal cortex. Overall, the aged macaques may be useful as a potential presymptomatic model for PD and, also, for other types neuropsychiatric disorders such as PDD, DLB, and AD.


The model that combined the selected metabolites with clinical parameters showed a significant larger AUC-ROC than that of the model containing only clinical parameters (p = 0.01). The combined model was able to discriminate good- and non-responders with good accuracy and to reclassify non-responders with an improvement of 30%  and showed a prediction error of 0.27. For the complete TNFi cohort, the NRI was 0.22. In addition, 88 metabolites were associated with DAS28, ESR or CRP (p<0.05). Our study established an accurate prediction model for response to TNFi therapy, containing metabolites and clinical parameters. Associations between metabolites and disease activity may help elucidate additional pathologic mechanisms behind RA.In clinical practice, approximately one-third of patients with rheumatoid arthritis (RA) respond insufficiently to TNF-α inhibitors (TNFis). The aim of the study was to explore the use of a metabolomics to identify predictors for the outcome of TNFi therapy, and study the metabolomic fingerprint in active RA irrespective of patients’ response. In the metabolomic profiling, lipids, oxylipins, and amines were measured in serum samples of RA patients from the observational BiOCURA cohort, before start of biological treatment. Multivariable logistic regression models were established to identify predictors for good- and non-response in patients receiving TNFi (n = 124). The added value of metabolites over prediction using clinical parameters only was determined by comparing the area under receiver operating characteristic curve (AUC-ROC), sensitivity, specificity, positive- and negative predictive value and by the net reclassification index (NRI). The models were further validated by 10-fold cross validation and tested on the complete TNFi treatment cohort including moderate responders. Additionally, metabolites were identified that cross-sectionally associated with the RA disease activity score based on a 28-joint count (DAS28), erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP). Out of 139 metabolites, the best-performing predictors were  Rheumatoid arthritis (RA) is a chronic, disabling disease that mainly affects the synovial joints. The disease is a multifactorial autoimmune disorder with a prevalence of 0.5–1% in industrialized countries . DiseaseMetabolomics is a rapidly developing approach in biomarker research, involving the measurement of a large number of small-molecule metabolites in biological fluids, tissues and cells. One major advantage is that it offers a characteristic profile of each patient from minimal amounts of sample. With high-throughput techniques, such as nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled to mass spectrometry (LC-MS), metabolite profiles in disease or therapeutic response to treatment can be measured . In this1H-NMR-based technique. Kapoor et al. . A. Ade novsn1-LPC (18:3-ω3/ω6) was found in good responders, while a down-regulated sn1-LPC (15:0) was found in non-responders. The most common structure of LPCs is with an even-chain fatty acid on sn1 or sn2 position, such as sn1-LPC (18:3-ω3/ω6) with an 18-carbon fatty acid. However, sn1-LPC (15:0) carries an odd-saturated fatty acid (OCS-FA) chain. As part of lipid metabolism, LPCs and FAs can interconvert , w, wsn1-LPyl group .In our study, several amines were found to be significantly associated with DAS28 (6 out of 7 negatively), CRP  and ESR (4 out of 5 positively). Of those amines negatively related with DAS28, histidine, asparagine, and threonine were reported to be downregulated in RA patients compared to healthy controls , 10]. I. I10]. IAlthough DAS28 has been extensively validated and is the most widely used instrument in clinical trials as well as in daily practice, it should also be noted that it has been claimed to be a rather instable monitoring instrument in RA patients with stable disease ,46. Two In this study, only metabolites at baseline were used to predict response and cross-sectionally investigate biomarkers for disease activity. As for the latter aim, investigating the change over time of metabolites might gain more insight in the most relevant metabolites regarding disease activity than any cross-sectional study, as they reflect prospective changes in RA patients.Metabolomic profiling is a powerful technique, which can be applied to analyze a wide range of metabolites from small sample volumes. Therefore, it has the potential of identification of biomarkers and increasing the understanding of the metabolic pathogenic pathways involved in a disease, such as RA. In the present study, we employed metabolic profiling to identify candidate metabolites to predict clinical response and assessed associations between metabolites and disease activity of RA. Because subjects were selected from an observational study, the heterogeneity needed to be adjusted for possible influential factors in all analyses. Yet, regardless of the possible absence of important influential factors, we showed that the predictive ability of a model can be quite high (AUC > 0.8) in a heterogeneous setting like clinical practice. As for its potential use, metabolites allow a better identification of non-responders on top of clinical parameters, which is of added value in determining the most fitting treatment for each individual patient. Further external validation is needed to assess the robustness of these findings and its potential value for clinical application.This is the first time that serum metabolomic profiles analyzed by LC-MS have been demonstrated to predict therapeutic response to biological treatment in RA. It would be worth studying how these metabolites could be used for predicting patients’ response with external validation in a more homogenous cohort and thereby potentially optimize the treatment strategy for patients with RA.S1 FigPatients were groups according to their observed responses on x-axis; y-axis represents the predicted probability calculated by the regression. The pseudo R-square, as a measure for the explained variance in the observed response by the model was 0.147 (COX & Snell).(TIF)Click here for additional data file.S2 FigPatients were groups according to their observed responses on x-axis; y-axis represents the predicted probability calculated by the regression. The pseudo R-square, as a measure for the explained variance in the observed response by the model was 0.433 (COX & Snell).(TIF)Click here for additional data file.S3 FigThe AUC-ROC was 0.641 (95% CI: 0.548–0.734).(TIF)Click here for additional data file.S4 FigThe AUC-ROC was 0.760 (95% CI: 0.682–0.837).(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.